Molecular docking study on the α3β2 neuronal nicotinic acetylcholine receptor complexed with α-conotoxin GIC

Nicotinic acetylcholine receptors (nAChRs) are a diverse family of homo- or heteropentameric ligand-gated ion channels. Understanding the physiological role of each nAChR subtype and the key residues responsible for normal and pathological states is important. α-Conotoxin neuropeptides are highly selective probes capable of discriminating different subtypes of nAChRs. In this study, we performed homology modeling to generate the neuronal α3, β2 and β4 subunits using the x-ray structure of the α1 subunit as a template. The structures of the extracellular domains containing ligand binding sites in the α3β2 and α3β4 nAChR subtypes were constructed using MD simulations and ligand docking processes in their free and ligand-bound states using α-conotoxin GIC, which exhibited the highest α3β2 vs. α3β4 discrimination ratio. The results provide a reasonable structural basis for such a discriminatory ability, supporting the idea that the present strategy can be used for future investigations on nAChR-ligand complexes.     

Diazaspirocyclic compounds as selective ligands for the α4β2 nicotinic acetylcholine receptor

Diazaspirocyclic ligands have been synthesized in four steps as selective α4β2 nicotinic acetylcholine receptor antagonists. Structural assignment of 1-(pyridin-3-yl)-2-spiropyrrolidino-3,2'-1-azabiclo[2.2.1]heptane 2, was confirmed using a combination of NMR experiments on a key intermediate, spirolactam 9. All three target compounds synthesized in this diazaspirocyclic series exhibited high affinity (K(i)<35 nM) at the human α4β2 nAChR subtype, and very low affinity for the human α7, α3β4 (ganglion) and α1β1γδ (muscle) subtypes (K(i)>500 nM).     

The α7 nicotinic acetylcholine receptor in neuronal plasticity

A growing body of evidence indicates that neuronal nicotinic acetylcholine receptors (nAChRs), in addition to promoting fast cholinergic transmission, may modulate other neuronal activities within the central nervous system (CNS). In particular, the α7 nAChR is highly permeable to Ca²⁺ and may serve a distinct role in regulating neuronal plasticity. By elevating intracellular Ca²⁺ levels in discrete neuronal locations, these ligand-gated ion channels may influence numerous physiological processes in developing and adult CNS. In this article, we review evidence that both pre- and postsynaptic α7 nAChRs modulate transmitter release in the brain and periphery through Ca²⁺-dependent mechanisms. The possible role of α7 nAChRs in regulating neuronal growth and differentiation in developing CNS is also evaluated. We consider an interaction between cholinergic and glutamatergic transmission and propose a hypothesis on the possible coregulation of intracellular Ca²⁺ byN-methyl-d-aspartate (NMDA) receptors and α7 nAChRs. Finally, the clinical significance of alterations in the normal function of α7 nAChRs is discussed as it pertains to prenatal nicotine exposure, schizophrenia, and epilepsy.     

Pharmacological and molecular studies on the interaction of varenicline with different nicotinic acetylcholine receptor subtypes. Potential mechanism underlying partial agonism at human α4β2 and α3β4 subtypes

To determine the structural components underlying differences in affinity, potency, and selectivity of varenicline for several human (h) nicotinic acetylcholine receptors (nAChRs), functional and structural experiments were performed. The Ca^2 + influx results established that: (a) varenicline activates (μM range) nAChR subtypes with the following rank sequence: hα7 > hα4β4 > hα4β2 > hα3β4 >>> hα1β1γδ; (b) varenicline binds to nAChR subtypes with the following affinity order (nM range): hα4β2 ~ hα4β4 > hα3β4 > hα7 >>> Torpedo α1β1γδ. The molecular docking results indicating that more hydrogen bond interactions are apparent for α4-containing nAChRs in comparison to other nAChRs may explain the observed higher affinity; and that (c) varenicline is a full agonist at hα7 (101%) and hα4β4 (93%), and a partial agonist at hα4β2 (20%) and hα3β4 (45%), relative to (±)-epibatidine. The allosteric sites found at the extracellular domain (EXD) of hα3β4 and hα4β2 nAChRs could explain the partial agonistic activity of varenicline on these nAChR subtypes. Molecular dynamics simulations show that the interaction of varenicline to each allosteric site decreases the capping of Loop C at the hα4β2 nAChR, suggesting that these allosteric interactions limit the initial step in the gating process. In conclusion, we propose that in addition to hα4β2 nAChRs, hα4β4 nAChRs can be considered as potential targets for the clinical activity of varenicline, and that the allosteric interactions at the hα3β4- and hα4β2-EXDs are alternative mechanisms underlying partial agonism at these nAChRs.     

Is agonist self-inhibition at the nicotinic acetylcholine receptor a nonspecific action?

Agonist concentration-response relationships at nicotinic postsynaptic receptors were established by measuring 86Rb+ efflux from acetylcholine receptor rich native Torpedo membrane vesicles under three different conditions: integrated net ion efflux (in 10 s) from untreated vesicles, integrated net efflux from vesicles in which most acetylcholine sites were irreversibly blocked with alpha-bungarotoxin, and initial rates of efflux (5-100 ms) from vesicles that were partially blocked with alpha-bungarotoxin. Exposure to acetylcholine, carbamylcholine, suberyldicholine, phenyltrimethylammonium, or (-)-nicotine over 10(8)-fold concentration ranges results in bell-shaped ion flux response curves due to stimulation of acetylcholine receptor channel opening at low concentrations and inhibition of channel function at 60-2000 times higher concentrations. Concentrations of agonists that inhibit their own maximum 86Rb+ efflux by 50% (KB values) are 110, 211, 3.0, 39, and 8.9 mM, respectively, for the agonists listed above. For acetylcholine and carbamylcholine, KB values determined from both 10-s and 15-ms efflux measurements are the same, indicating that the rate of agonist-induced desensitization increases to maximum at concentrations lower than those causing self-inhibition. For all partial and full agonists studied, Hill coefficients for self-inhibition are close to 1.0. Concentrations of agonists up to 8 times KB did not change the order parameter reported by a spin-labeled fatty acid incorporated in Torpedo membranes. We conclude that agonist self-inhibition cannot be attributed to a general nonspecific membrane perturbation. Instead, these results are consistent with a saturable site of action either at the lipid-protein interface or on the acetylcholine receptor protein itself.     

Computational neural network analysis of the affinity of N-n-alkylnicotinium salts for the α4β2* nicotinic acetylcholine receptor

Based on an 85 molecule database, linear regression with different size datasets and an artificial neural network approach have been used to build mathematical relationships to fit experimentally obtained affinity values (K_i) of a series of mono- and bis-quaternary ammonium salts from [³H]nicotine binding assays using rat striatal membrane preparations. The fitted results were then used to analyze the pattern among the experimental K_i values of a set of N-n-alkylnicotinium analogs with increasing n-alkyl chain length from 1 to 20 carbons. The affinity of these N-n-alkylnicotinium compounds was shown to parabolically vary with increasing numbers of carbon atoms in the n-alkyl chain, with a local minimum for the C₄ (n-butyl) analogue. A decrease in K_i value between C₁₂ and C₁₃ was also observed. The statistical results for the best neural network fit of the 85 experimental K_i values are r² = 0.84, rmsd = 0.39; r_cv² = 0.68, and loormsd = 0.56. The generated neural network model with the 85 molecule training set may also be of value for future predictions of K_i values for new virtual compounds, which can then be identified, subsequently synthesized, and tested experimentally.     

Polymorphisms in the neural nicotinic acetylcholine receptor α4 subunit (CHRNA4) are associated with ADHD in a genetic isolate

The neural nicotinic acetylcholine receptor α4 subunit (CHRNA4), at 20q13.2-q13.3, is an important candidate gene for conferring susceptibility to attention deficit/hyperactivity disorder (ADHD). Several studies have already looked for association/linkage between ADHD and CHRNA4 in different populations. We used the Pedigree Disequilibrium Test to search for evidence of association between ADHD and six SNP marker loci in families from the isolated Paisa population. We found that the T allele of SNP rs6090384 exhibits a deficit of transmission in unaffected individuals (OR = 5.43, IC 1.54-19.13) (global P value = 0.014). We also found significant association and linkage to extended haplotypes rs2273502-rs6090384 (combination of variants C-T, respectively) (P = 0.02) and rs6090384-rs6090387 (P = 0.04) (combination of variants T-G, respectively). SNP rs6090384, variant T, has also been reported to be associated with inattention in a previous study. This makes ours the ninth study to examine the association of CHRNA4 with ADHD and the seventh one to find evidence for association in a population with a different ethnicity.     

Metabotropic signaling cascade involved in α4β2 nicotinic acetylcholine receptor-mediated PKCβII activation

Nicotinic acetylcholine receptors (nAChRs) belong to the ionophore receptor family, which regulates plasma membrane conductance to Na⁺, K⁺, and Ca²⁺ ions. Some studies, however, have shown that nAChRs also employ second messengers for intracellular signaling. We previously showed that α4β2 nAChR mediates the translocation of protein kinase CβII (PKCβII) from the cytoplasm to the plasma membrane, which is a typical activation marker for PKCβII. In this study, we investigated the molecular mechanisms underlying PKCβII activation through α4β2 nAChR. α4β2 nAChR is the most abundant nAChR subtype and is implicated in various brain functions and diseases. Putative α4β2 nAChR signaling components were identified by knockdown or chemical inhibition of candidate proteins, and the signaling cascade was deduced by protein interactions in predicted cellular components. α4β2 nAChR-mediated PKCβII translocation was found to occur in an ionophore activity-independent manner. Nicotinic stimulation of α4β2 nAChR activated Src in a β-arrestin1 and 14–3-3η-dependent manner. Activated Src phosphorylated the tyrosine residue(s) on Syk molecules, which in turn interacted with phospholipase C γ1 to trigger the translocation of PKCβII to the cell membrane by elevating cellular diacylglycerol levels. The activated PKCβII in turn exerted a positive feedback effect on Src activation, suggesting that α4β2 nAChR signaling is amplified by a positive feedback loop. These findings provide novel information for unveiling the previously unclear metabotropic second messenger-based signal transduction pathway of nAChRs.     

Multiple nicotinic acetylcholine receptor α-subunits are expressed in the arterial system of the rat

Neuronal nicotinic acetylcholine receptors (nAChRs) are hetero- and homopentamers built up by nine different alpha-subunits and three different beta-subunits. The subtype composition within the receptor determines ligand specificity, affinity and cation permeability. In this study we focused on the distribution of the ligand binding alpha-subunits in the rat arterial system by means of RT-PCR and immunohistochemistry. Subtypes alpha3, alpha5, alpha7 and alpha10 were found to be expressed by endothelial cells, suggesting that they are equipped both with calcium-preferring (alpha7 homopentamers) and monovalent cation-preferring (heteropentamers containing alpha3- and alpha5-subunits) nAChR channels. All alpha-subtypes except alpha9 were expressed by vascular smooth muscle cells with a highly specific distribution pattern along the vascular tree. While every alpha-subunit except alpha9 was detected in the thoracic aorta, intrapulmonary arterial branches contained only alpha7 immunoreactivity, and other vascular beds held intermediate positions with respect to the extent of alpha-subunit expression. Current knowledge does not allow to correlate these distribution patterns to specific functions, but it can be anticipated that at least some components of nAChR-mediated signalling in the arterial wall are highly specific for individual arteries.     

Photoaffinity labeling the agonist binding domain of α4β4 and α4β2 neuronal nicotinic acetylcholine receptors with [I]epibatidine and 5[I]A-85380

The development of nicotinic acetylcholine receptor (nAChR) agonists, particularly those that discriminate between neuronal nAChR subtypes, holds promise as potential therapeutic agents for many neurological diseases and disorders. To this end, we photoaffinity labeled human α4β2 and rat α4β4 nAChRs affinity-purified from stably transfected HEK-293 cells, with the agonists [¹²⁵I]epibatidine and 5[¹²⁵I]A-85380. Our results show that both agonists photoincorporated into the β4 subunit with little or no labeling of the β2 and α4 subunits respectively. [¹²⁵I]epibatidine labeling in the β4 subunit was mapped to two overlapping proteolytic fragments that begin at β4V102 and contain Loop E (β4I109-P120) of the agonist binding site. We were unable to identify labeled amino acid(s) in Loop E by protein sequencing, but we were able to demonstrate that β4Q117 in Loop E is the principal site of [¹²⁵I]epibatidine labeling. This was accomplished by substituting residues in the β2 subunit with the β4 homologs and finding [¹²⁵I]epibatidine labeling in β4 and β2F119Q subunits with little, if any, labeling in α4, β2, or β2S113R subunits. Finally, functional studies established that the β2F119/β4Q117 position is an important determinant of the receptor subtype-selectivity of the agonist 5I-A-85380, affecting both binding affinity and channel activation.     

Molecular modeling of the α9α10 nicotinic acetylcholine receptor subtype

This study reports the comparative molecular modeling, docking and dynamic simulations of human α9α10 nicotinic acetylcholine receptors complexed with acetylcholine, nicotine and α-conotoxin RgIA, using as templates the crystal structures of Aplysia californica and Lymnaea stagnalis acetylcholine binding proteins. The molecular dynamics simulations showed that Arg112 in the complementary α10(?) subunit, is a determinant for recognition in the site that binds small ligands. However, Glu195 in the principal α9(+), and Asp114 in the complementary α10(?) subunit, might confer the potency and selectivity to α-conotoxin RgIA when interacting with Arg7 and Arg9 of this ligand.     

Novel positive allosteric modulators of the human α7 nicotinic acetylcholine receptor

The pharmacological activity of a series of novel amide derivatives was characterized on several nicotinic acetylcholine receptors (AChRs). Ca(2+) influx results indicate that these compounds are not agonists of the human (h) α4β2, α3β4, α7, and α1β1γδ AChRs; compounds 2-4 are specific positive allosteric modulators (PAMs) of hα7 AChRs, whereas compounds 1-4, 7, and 12 are noncompetitive antagonists of the other AChRs. Radioligand binding results indicate that PAMs do not inhibit binding of [(3)H]methyllycaconitine but enhance binding of [(3)H]epibatidine to hα7 AChRs, indicating that these compounds do not directly, but allosterically, interact with the hα7 agonist sites. Additional competition binding results indicate that the antagonistic action mediated by these compounds is produced by direct interaction with neither the phencyclidine site in the Torpedo AChR ion channel nor the imipramine and the agonist sites in the hα4β2 and hα3β4 AChRs. Molecular dynamics and docking results suggest that the binding site for compounds 2-4 is mainly located in the inner β-sheet of the hα7-α7 interface, ～12 ? from the agonist locus. Hydrogen bond interactions between the amide group of the PAMs and the hα7 AChR binding site are found to be critical for their activity. The dual PAM and antagonistic activities elicited by compounds 2-4 might be therapeutically important.     

Neonicotinic analogues: Selective antagonists for α4β2 nicotinic acetylcholine receptors

Nicotine is an agonist of nicotinic acetylcholine receptors (nAChRs) that has been extensively used as a template for the synthesis of α4β2-preferring nAChRs. Here, we used the N-methyl-pyrrolidine moiety of nicotine to design and synthesise novel α4β2-preferring neonicotinic ligands. We increased the distance between the basic nitrogen and aromatic group of nicotine by introducing an ester functionality that also mimics acetylcholine (Fig. 2). Additionally, we introduced a benzyloxy group linked to the benzoyl moiety. Although the neonicotinic compounds fully inhibited binding of both [α-¹²⁵I]bungarotoxin to human α7 nAChRs and [³H]cytisine to human α4β2 nAChRs, they were markedly more potent at displacing radioligand binding to human α4β2 nAChRs than to α7 nAChRs. Functional assays showed that the neonicotinic compounds behave as antagonists at α4β2 and α4β2α5 nAChRs. Substitutions on the aromatic ring of the compounds produced compounds that displayed marked selectivity for α4β2 or α4β2α5 nAChRs. Docking of the compounds on homology models of the agonist binding site at the α4/β2 subunit interfaces of α4β2 nAChRs suggested the compounds inhibit function of this nAChR type by binding the agonist binding site.     

Structural characterization and agonist binding to human α4β2 nicotinic receptors

The Cys-loop receptor super-family of neurotransmitter-gated ion channels mediates fast synaptic transmission throughout the human nervous system. These receptors exhibit widely varying pharmacologies, yet their structural characterization has relied heavily on their homology with the naturally abundant muscle-type Torpedo nicotinic acetylcholine receptor. Here we examine for the first time the structure of a human α4β2 neuronal nicotinic acetylcholine receptor. We show that human α4β2 nicotinic receptors adopt a secondary/tertiary fold similar to that of the Torpedo nicotinic receptor with a large proportion of both α-helix and β-sheet, but exhibit a substantially increased thermal stability. Both receptors bind agonist, but with different patterns of agonist recognition – particularly in the nature of the interactions between aromatic residues and the agonist quaternary amine functional group. By comparing α4β2 and Torpedo receptors, we begin to delineate their structural similarities and differences.     

Changes in temperature have opposing effects on current amplitude in α7 and α4β2 nicotinic acetylcholine receptors

We have examined the effect of temperature on the electrophysiological properties of three neuronal nicotinic acetylcholine receptor (nAChR) subtypes: the rapidly desensitizing homomeric α7 nAChR, the more slowly desensitizing heteromeric α4β2 nAChR and on α7 nAChRs containing a transmembrane mutation (L247T) that results in dramatically reduced desensitization. In all cases, the functional properties of receptors expressed in Xenopus oocytes at room temperature (RT; 21°C) were compared to those recorded at either physiological temperature (37°C) or at lower temperature (4°C). Alterations in temperature had dramatically differing effects on the amplitude of whole-cell responses detected with these three nAChR subtypes. Compared to responses at RT, the amplitude of agonist-evoked responses with α4β2 nAChRs was increased at high temperature (125±9%, n = 6, P<0.01) and reduced at low temperature (47±5%, n = 6, P<0.01), whereas the amplitude of α7 responses was reduced at high temperature (27±7%, n = 11, P<0.001) and increased at low temperatures (224±16%, n = 10, P<0.001). In contrast to the effects of temperature on α4β2 and wild type α7 nAChRs, the amplitude of α7 nAChRs containing the L247T mutation was unaffected by changes in temperature. In addition, changes in temperature had little or no effect on current amplitude when α7 nAChRs were activated by the largely non-desensitizing allosteric agonist 4BP-TQS. Despite these differing effects of temperature on the amplitude of agonist-evoked responses in different nAChRs, changes in temperature had a consistent effect on the rate of receptor desensitization on all subtypes examined. In all cases, higher temperature resulted in increased rates of desensitization. Thus, it appears that the differing effects of temperature on the amplitudes of whole-cell responses cannot be explained by temperature-induced changes in receptor desensitization rates.     

Homology modeling and dynamics of the extracellular domain of rat and human neuronal nicotinic acetylcholine receptor subtypes α4β2 and α7

In recent years, it has become clear that the neuronal nicotinic acetylcholine receptor (nAChR) is a valid target in the treatment of a variety of diseases, including Alzheimer’s disease, anxiety, and nicotine addiction. As with most membrane proteins, information on the three-dimensional (3D) structure of nAChR is limited to data from electron microscopy, at a resolution that makes the application of structure-based design approaches to develop specific ligands difficult. Based on a high-resolution crystal structure of AChBP, homology models of the extracellular domain of the neuronal rat and human nAChR subtypes α4β2 and α7 (the subtypes most abundant in brain) were built, and their stability assessed with molecular dynamics (MD). All models built showed conformational stability over time, confirming the quality of the starting 3D model. Lipophilicity and electrostatic potential studies performed on the rat and human α4β2 and α7 nicotinic models were compared to AChBP, revealing the importance of the hydrophobic aromatic pocket and the critical role of the α-subunit Trp—the homolog of AChBP-Trp 143—for ligand binding. The models presented provide a valuable framework for the structure-based design of specific α4β2 nAChR subtype ligands aimed at improving therapeutic and diagnostic applications. Figure Electrostatic surface potential of the binding site cavity of the neuronal nicotinic acetylcholine receptor (nAChR). Nicotinic models performed with the MOLCAD program: a rat α7, b rat α4β2, c human α7, d human α4β2. All residues labeled are part of the α7 (a,c) or α4 (b,d) subunit with the exception of Phe 117, which belongs to subunit β2 (d). Violet Very negative, blue negative, yellow neutral, red very positive     

Inhibition mechanism of rat α₃β₄ nicotinic acetylcholine receptor by the Alzheimer therapeutic tacrine

Nicotinic acetylcholine receptors (nAChRs) were studied in detail in the past regarding their interaction with therapeutic and drug addiction related compounds. Using fast kinetic whole-cell recording, we have now studied effects of tacrine, an agent used clinically to treat Alzheimer's disease, on currents elicited by activation of rat α(3)β(4) nAChR heterologously expressed in KXα3β4R2 cells. Characterization of receptor activation by nicotine used as agonist revealed a K(d) of 23 ± 0.2 μM and 4.3 ± 1.3 for the channel opening equilibrium constant, Φ(-1). Experiments were performed to investigate whether tacrine is able to activate the α(3)β(4) nAChR. Tacrine did not activate whole-cell currents in KXα3β4R2 cells but inhibited receptor activity at submicromolar concentration. Dose-response curves obtained with increasing agonist or inhibitor concentration revealed competitive inhibition of nAChRs by tacrine, with an apparent inhibition constant, K(I), of 0.8 μM. The increase of Φ(-1) in the presence of tacrine suggests that the drug stabilizes a nonconducting open channel form of the receptor. Binding studies with TCP and MK-801 ruled out tacrine binding to common allosteric sites of the receptor. Our study suggests a novel mechanism for action of tacrine on nAChRs besides inhibition of acetylcholine esterase.